Like all herpesviruses, HSV-1 consists of an icosahedral capsid surrounded by a membrane envelope. The capsid, which contains the virus DNA, is assembled in the infected cell nucleus. A DNA-free capsid shell is first formed and later packaged with DNA. Capsids are assembled from a major structural protein (UL19), three other structural polypeptides and a scaffolding protein. Capsids must be formed in such a way that they are able to release the encapsidated DNA as a new cycle of infection is initiated. Individual steps in capsid assembly and DNA uncoating are regarded as attractive targets for novel therapeutics because they are required for HSV-1 replication and because virus-encoded proteins are the primary components involved. In their basic features, the steps of capsid assembly and DNA egress in HSV-1 are expected to be the same as those for other herpesviruses. Thus potential drug targets identified in HSV-1 should be able to be exploited for the design of therapeutics effective against other herpesviruses including human cytomegalovirus, Epstein- Barr virus and Kaposi's sarcoma-associated herpesvirus. A new initiative is proposed to examine how DNA is released from the capsid to initiate an infection. DNA release or uncoating will be studied beginning with purified DNA-containing capsids and an in vitro uncoating system recently developed in our laboratory. Experiments will be carried out to identify the DNA end that emerges first from the capsid and to clarify the nature of heterogeneity observed in the population of DNA-containing capsids (aim 1). In vitro studies will be complemented with an analysis of DNA uncoating as it occurs in infected cells. This project will focus on identifying protein contacts between the capsid and the nuclear pore, the organelle through which DNA enters the host cell nucleus (aim 2). Other proposed studies will make use of a cell-free capsid assembly system to examine the way the portal becomes incorporated into the nascent capsid. We propose to test the hypothesis that assembly involves a filamentous aggregate of the scaffolding protein bound to the portal and major capsid proteins (aim 3). This is proposed to condense in a stepwise fashion to create the procapsid. Studies will also be performed to identify how components of the DNA packaging machinery are assembled on the capsid surface prior to their functioning in DNA translocation (aim 4). PUBLIC HEALTH RELEVANCE: Herpes simplex virus (HSV-1) continues to be the cause of widespread human illness despite the availability of a reasonably effective therapeutic, acyclovir and related compounds. Diseases caused by HSV-1 include disseminated illness in the newborn, cold sores, genital lesions, non-epidemic encephalitis in adults, a stromal keratitis and retinitis. The goal of the proposed research is to study assembly of the virus capsid with the aim of identifying novel targets against which small molecule inhibitors might be directed.